Rapid separation of peptides and proteins by isocratic capillary electrochromatography at elevated temperature

The use of capillary electrochromatography (CEC) for the separation by isoc ratic elution of synthetic peptides, proteins as well as the tryptic digest of cytochrome c has been demonstrated. The monolithic porous stationary ph ase was prepared from silanized fused-silica capillaries of 75 mum I.D, by in situ copolymerization of vinylbenzyl chloride and ethylene glycol dimeth acrylate in the presence of propanol and formamide as the porogens. The chl oromethyl groups at the surface of the porous monolith were reacted with N, N-dimethylbutylamine to form a positively charged chromatographic surface w ith fixed n-butyl chains. Results of studies on the influence of temperatur e and mobile phase composition on the retention and selectivity of separati on by CEC demonstrated the feasibility of rapid polypeptide analysis and tr yptic mapping at elevated temperature with high resolution and efficiency. Typically the chromatography of a tryptic digest of cytochrome c took about 5 min at 55 degreesC and 75 kV/m with hydro-organic mobile phases containi ng acetonitrile in 50 mM phosphate buffer, pH 2.5. For peptides and protein s plots of logarithmic kf,, against acetonitrile concentration were nonline ar, whereas Arrhenius plots for the mobilities were nearly linear. Comparis on of the separation of such samples under conditions of CEC and capillary zone electrophoresis (CZE) indicates that the mechanism of separation in CE C is unique and leads to a chromatographic profile different from that obta ined by CZE. (C) 2001 Elsevier Science B.V. All rights reserved.